Systems and methods for cell culturing

ABSTRACT

Cell culture systems and methods provide improved immunotherapeutic product manufacturing with greater scalability, flexibility, and automation. Cell culture systems are configured with interchangeable cartridges, allowing versatility and scalability. Systems are configured to have multiple connected cell culture chambers, which allows parallel processing of different types of cells. Gas-impermeable cell culture chambers and methods for generating cells in closed systems prevent contamination and user error. Methods for recycling cell culture medium provide additional efficiencies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to, and the benefit of, U.S.Provisional Application No. 62/923,963, filed Oct. 21, 2019, and to U.S.Provisional Application No. 62/923,967, filed Oct. 21, 2019, and to U.S.Provisional Application No. 62/923,973, filed Oct. 21, 2019, and to U.S.Provisional Application No. 62/923,975, filed Oct. 21, 2019, and to U.S.Provisional Application No. 62/923,978, filed Oct. 21, 2019, and to U.S.Provisional Application No. 62/923,982, filed Oct. 21, 2019, thecontents of which are incorporated by reference herein in theirentirety.

FIELD OF THE INVENTION

The present disclosure relates generally to systems and methods for cellculturing.

BACKGROUND

Cancer is a leading cause of mortality and morbidity worldwide, anddespite years of extraordinary research efforts, treatments haveremained elusive. The diversity of tumor types presents a challenge incancer therapy, as treatments tailored to one tumor may not be effectiveagainst another. Personalized treatments have been sought, but manychallenges exist in developing them.

One promising area has been T cell therapy, wherein a patient's T cellsare altered to target certain cancers. This includes chimeric antigenreceptor T cell (CAR-T) therapy, T cell receptor (TCR) therapy, andneoantigen-based T cell therapy. Neoantigen-based therapies provide theability to identify antigens from tumor sequencing data to design highlypersonalized patient-specific immunotherapies.

Unfortunately, many challenges exist in the development and manufactureof T cell therapies. Existing processes for isolation, preparation, andexpansion of cancer antigen-specific T-cells are limited. Conventionalprotocols for stimulation of human T cells by autologousantigen-presenting dendritic cells (DCs) involve several manual steps,including transferring cells between culture vessels, changing media,and replenishing cytokines and cell medium. Those processes arelabor-intensive and not readily scalable. The number of manual stepsrequired to carry out the protocol is prohibitively high. Additionally,those protocols involve the use of flasks or other containers, which areopened and closed during use, adding to the risk of contamination whichcan compromise the quality and safety of the cell product. Such methodsdo not comply with current good manufacturing practices (cGMP) and arenot useful for producing T cell therapies at large scale. Additionalchallenges also exist, such as time of cell preparation, maintenance ofoptimal phenotype, expansion to sufficient cell number, and quality andsafety of the cell product.

SUMMARY

The invention recognizes that automating T cell therapy processing andmanufacturing has been unsuccessful due to the complex biologicalprocesses associated as well as the bioprocess and regulatoryrequirements associated with autologous cell processing. The few systemsthat do exist are overly complex and cost-prohibitive, and are thereforeare not useful for pre-clinical assays. The inventive cell culturesystems and related methods of the invention provide solutions to manyof the problems in cell culturing and provides numerous features todecrease contamination and user error, as well as increase efficiency,scalability, and ease of use. The systems and methods of the inventionprovide capabilities for robust T cell production, while minimizing costand increasing simplicity and ease of use, making the disclosed systemsand methods useful for both pre-clinical research and routine cellculture, while being capable of meeting requirements for current goodmanufacturing practices for clinical manufacturing.

In certain aspects, the disclosed systems and methods provide improvedautomated technology for producing antigen-specific T cells. Automationof the manual processes dramatically reduces opportunities for usererror and decreases the risk of contamination. For example, thedisclosure provides systems and methods for producing CAR-T and TCRtransduced T cells, as well as neoantigen-targeting T cells in a closedsystem. This avoids the need to open and close T flasks, as is common inthe prior art, thereby simplifying the process and avoiding sources ofcontamination. As another example, cell culture systems are disclosedwhich are configured with easily interchangeable cell culture chambers,allowing the user to scale up or scale down a cell population. Thevarious chambers and vessels are connectable via sterile tube welding,so that the system can remain closed throughout use. The disclosure alsoprovides gas-impermeable cartridges for cell culture, which provide asolid polystyrene surface for optimal cell adhesion and rigid cartridgeconstruction which is easy to manufacture and less susceptible tocontamination when operated with welded tube connections. The disclosedsystems also allow parallel processing of dendritic cells and T cells ina process for generating stimulated T cells. The system architecturestreamlines the process of T cell culture, providing savings in time andmaterials. Another way that the system saves materials is by recyclingcell culture medium, to ensure that cells can be cultured with minimalamount of expensive culture medium and supplements.

In addition to the features described above, other features will beapparent to those of skill in the art, as the disclosed systems andmethods provide numerous opportunities for process optimization inimmunotherapeutic product manufacturing.

Aspects of the disclosure provide a cell culture system withinterchangeable cartridges. The cell culture system includes a firstarea configured to receive a fluid reservoir containing a cell culturemedium and a second area configured to receive a waste reservoir. Thecell culture system also has one or more pumps fluidically connectableto the fluid reservoir and a substrate configured to receive and retaincell culture chambers of different shapes and/or sizes.

In embodiments, the substrate has a plurality of different openingsarranged such that the substrate is configured to receive and retaincell culture chambers of different shapes and/or sizes. The substratecan be configured to receive and retain multiple cell culture chamberssimultaneously. A first portion of the substrate may be configured toreceive a first cell culture chamber of a first size whereas a secondportion of the substrate is configured to receive a second cell culturechamber of a second shape which is different from the first size. Inembodiments, the first portion of the substrate is configured to receivea first cell culture chamber of a first shape and the second portion ofthe substrate is configured to receive a second cell culture chamber ofa second shape that is different from the first shape. In embodiments,the first portion of the substrate is configured to receive a first cellculture chamber of a first size and shape and a second portion of thesubstrate is configured to receive a second cell culture chamber of asecond size and shape that is different from the first size and shape.

In embodiments, the fluid reservoir is positioned in the first area andthe waste reservoir is positioned in the waste area. One or more tubescan be included that fluidically connect the fluid reservoir to the oneor more pumps, and/or the one or more pumps to the cell culturechambers, and/or the cell culture chambers to the waste reservoir. Eachcell culture chamber can be fluidically coupled to a separate pump. Insome embodiments, a processor is operably connected to the one or morepumps and one or more sensors are operable to measure a characteristicof a fluid in the cell culture system, wherein the processor operatesthe one or more pumps based on the measured characteristic.

In a related aspect, the disclosure provides a method for culturingcells. The method includes providing a cell culture system that has afirst area configured to receive a fluid reservoir containing a cellculture medium and a second area configured to receive a wastereservoir, one or more pumps fluidically connectable to the fluidreservoir, and a substrate configured to receive and retain cell culturechambers of different shapes and/or sizes. The method further involvesloading the fluid reservoir into the first area and the waste reservoirinto the second area, loading a first cell culture chamber of a firstsize and/or shape onto a first portion of the substrate, and loading asecond cell culture chamber of a second size and/or shape onto a secondportion of the substrate. The method further involves connecting thefluid reservoir, the one or more pumps, the first and second cellculture chambers, and the waste reservoir with tubing. The methodfurther involves operating the system to culture cells in the first andsecond cell culture chambers.

In embodiments, the substrate has a plurality of different openingsarranged such that the substrate is configured to receive and retaincell culture chambers of different shapes and/or sizes. The first cellculture chamber can be of a first size and the second cell culturechamber can be of a second size. The first cell culture chamber can beof a first shape and the second cell culture chamber can be of a secondshape. The first cell culture chamber can be of both a first size andshape, and the second cell culture chamber can be of both a second sizeand shape.

In embodiments, each of the first and second cell culture chambers isfluidically coupled to a separate pump. The system can also include aprocessor operably connected to the one or more pumps and one or moresensors operable to measure a characteristic of a fluid in the cellculture system, wherein the processor operates the one or more pumpsbased on the measured characteristic.

In embodiments, after cell culturing is complete in the first and secondcell culture chambers, the cultured cells in each of the first andsecond cell culture chambers are collected. The cultured cells in eachof the first and second cell culture chambers can be collected in thesame collection vessel or in different collection vessels.

In another aspect, the disclosure provides a method for producingtransduced T cells with CAR or TCR in a closed system. The methodinvolves providing a cell culture instrument that has first and secondculture chambers and flowing a suspension containing cells into thefirst culture chamber. The method further involves perfusing the T cellsin the first culture chamber with appropriate transduction and expansionreagents to produce transduced T cells which expand in the first culturechamber. The method further involves flowing the transduced and expandedT cells from the first culture chamber into the second culture chamber.The method further involves flowing a cell culture medium into thesecond culture chamber to further expand the tranduced and expanded Tcells, wherein the method is performed on a single instrument in aclosed manner such that sterility is maintained throughout the method.

In some embodiments, the second culture chamber is larger than the firstculture chamber. One or both culture chambers can be made ofpolystyrene. The culture chambers can be connected via a sterile tube.The first culture chamber may have an activation reagent and/or a celltransduction reagent, which may be an inactive virus expressing CAR orTCR. Alternatively the second culture chamber may be a separate cellculture instrument that is not part of the first cell cultureinstrument.

In embodiments, the cell culture medium is provided in a sterile vesseland is connected to the closed system by sterile tube welding. Flowingthe cell culture medium into the first culture chamber may involveeliminating headspace in the first culture chamber. The cell culturemedium may include Aim V with interleukin-2.

The method may further involve activating the T cells in the firstculture chamber, which can be done by contacting with a magnetic ornon-magnetic bead comprising one or more activating antibodies orsoluble activation antibody-containing reagents, and a transductionreagent. The method may further involve draining fluid from the secondculture chamber, washing the transduced and expanded T cells with abuffer, and flowing a cryopreservation medium into the second culturechamber to re-suspend the transduced and expanded T cells. the methodmay further involve flowing the transduced and expanded T-cells into aharvesting vessel in a closed manner.

In embodiments, each of the flowing steps may be done via sterile tubes.The sterile tubes may be connected by sterile tube welding.

In another aspect, the disclosure provides a method for producingneoantigen-targeting T cells in a closed system. The method includesproviding a cell culture instrument having first and second culturechambers and flowing cell culture medium containing monocytes into thefirst culture chamber. The method also involves perfusing the purifiedmonocytes in the first culture chamber to produce dendritic cells in thefirst culture chamber and contacting the dendritic cells with antigenmaterial, which may include tumor-specific peptides, in the firstculture chamber to produce mature dendritic cells. The method furtherinvolves flowing the mature dendritic cells from the first culturechamber into the second culture chamber comprising purified T cells toco-culture the mature dendritic cells and the purified T cells, tothereby produce neoantigen-targeting T cells. The method is performed ona single instrument in a closed manner such that sterility is maintainedthroughout the method.

In embodiments, the method also involves flowing a second batch ofmonocytes into the second culture chamber, differentiating them intodendritic cells and maturing the dendritic cells, in order to thenperform a second co-culture with the purified T cells. The first andsecond culture chambers can be made of polystyrene. The first and secondculture chambers can be connected via a sterile tube. The cell culturemedium can be provided in a sterile vessel and can be connected to theclosed system by sterile tube welding. The step of flowing the cellculture medium into the first culture chamber can involve eliminatingheadspace in the first culture chamber. Each of the flowing steps can bedone via sterile tubes, which may be connected by sterile tube welding.

In embodiments, the method also includes activating the T cells in thesecond culture chamber. In embodiments, the method also includesdraining fluid from the second culture chamber, washing theneoantigen-targeting T cells with a buffer, and flowing acryopreservation medium into the second culture chamber to re-suspendthe neoantigen-targeting T cells. The method may also involve flowingthe neoantigen-targeting T cells into a harvesting vessel in a closedmanner.

In another aspect, the disclosure provides a method for parallelprocessing to produce dendritic cells and stimulate T cells in parallel.The method includes providing a cell culture instrument with first andsecond culture chambers and flowing cell culture medium containingmonocytes into the first culture chamber. The method further includesperfusing the monocytes in the first culture chamber to producedendritic cells in the first culture chamber. The method furtherincludes flowing T cells that have been cultured in the second culturechamber from the second culture chamber into the first culture chamberwith the dendritic cells to further culture the T cells in the firstculture chamber. In embodiments, sterility is maintained throughout themethod.

The method may also include collecting the cultured T cells from thefirst culture chamber by flowing the cultured T cells into a collectionvessel. The method may also include maturing the dendritic cells in thefirst culture chamber by contacting the dendritic cells with antigenmaterial, which may include tumor-specific peptides. In embodiments, themethod also involves activating the T cells in the second culturechamber, which can be done by using an activation reagent. Inembodiments, the method also involves washing the stimulated T cellswith a buffer, and optionally transferring the stimulated T cells to acryopreservation medium. The method may also involve flowing theneoantigen-targeting T cells into a harvesting vessel in a closedmanner. Each of the flowing steps can be done via sterile tubes, whichare optionally connected by sterile tube welding.

The cell culture medium may be provided in a sterile vessel and may beconnected to the closed system by sterile tube welding. Flowing the cellculture medium into the first culture chamber may involve eliminatingheadspace in the first culture chamber. In embodiments, the first andsecond culture chambers are made of polystyrene, and optionally may beconnected via a sterile tube. In some embodiments, one or both of thefirst and second cell culture chambers from the cell culture instrumentcan be replaced and the method can be repeated.

In another aspect, the disclosure provides a gas-impermeable cellculture chamber, wherein a top, a bottom, and both side walls arecomprised of a gas-impermeable material. The gas-impermeable materialmay also be a material to which cells adhere. The gas-impermeablematerial may be polystyrene.

In embodiments, the cell culture chamber has an inlet. The cell culturechamber may also have an outlet. The inlet and the outlet can be locatedon the top of the cell culture chamber, and optionally the inlet and theoutlet are each configured to fluidically and sealably couple withtubing. The cell culture chamber can be integrally formed, and it can besized and configured to fit within an incubator. The cell culturechamber can be sized and configured to couple to a substrate of a cellculture instrument.

In a related aspect, the disclosure provides a method for culturingcells that involves providing a cell culture chamber having an inlet andan outlet, wherein a top, a bottom, and both side walls are made of agas-impermeable material. The method also involves loading cells in tothe cell culture chamber and flowing a cell culture medium into the cellculture chamber via the inlet to culture the cells in the culturechamber and out of the cell culture chamber via the outlet, wherein theflowing of the cell culture medium through the cell culture chamber viathe inlet and the outlet causes continuous flow of cell culture mediumthrough the cell culture chamber and allows for gas exchange to occurbetween the cells in the cell culture chamber and the cell culturemedium.

In embodiments, the gas-impermeable material is also a material to whichcells adhere, such as polystyrene. In embodiments, the inlet and theoutlet are located on the top of the cell culture chamber and areoptionally configured to fluidically and sealably couple with tubing.The tubing can be high permeability tubing which allows the cell culturemedium to exchange gas while in the high permeability tubing. Inembodiments, the cell culture chamber is integrally formed. The cellculture chamber can be sized and configured to fit within an incubatorand optionally it can be sized and configured to couple to a substrateof a cell culture instrument.

In another aspect, the disclosure provides a method for culturing cellsthat involves culturing cells in a cell culture chamber on a cellculture instrument by flowing a cell culture medium through the cellculture chamber, wherein a portion of the cell culture medium that hasalready been flowed through the cell culture chamber is recycled backinto the cell culture chamber during the cell culturing process.

In embodiments, the method also involves measuring one or moreparameters of the used medium prior to the recycling. The parameters canbe a concentration of one or more compounds within the used medium, suchas glucose, lactate, dissolved oxygen, or cell metabolites. Theparameter can also be pH or cell number.

In embodiments, the method involves determining, using a processoroperably connected to the cell culture chamber, whether at least one ofthe one or more parameters of the used medium meets a predeterminedthreshold prior to the recycling step. The measuring step can beperformed by one or more sensors operably associated with the cellculture chamber. The one or more sensors can be operably associated witha waste reservoir in fluid communication with the cell culture chamber.The cell culture chamber can be operably connected to one or more pumps,and may have an inlet and an outlet. The recycling step may involveredirecting the portion of used medium from the waste reservoir backinto the cell culture chamber. In embodiments, the portion of usedmedium is combined with a bolus of fresh medium.

In a related aspect, the disclosure provides a method for culturingcells, which involves providing a cell culture chamber containing cells,flowing a cell culture medium into the cell culture chamber, removingused cell culture medium from the cell culture chamber, assessing aparameter of the used cell culture medium, and returning the used cellculture medium to the cell culture chamber if the parameter meets apredetermined threshold.

In embodiments, the method also involves combining the used cell culturemedium with a bolus of fresh cell culture medium prior to the returningstep. The assessing step may involve measuring the parameter using asensor operably coupled to the cell culture chamber. The assessing stepmay involve determining, using a processor, whether the parameter meetsthe predetermined threshold. The parameter may be a measuredconcentration of one or more compounds within the used cell culturemedium, such as glucose, lactate, or cell metabolites. In embodiments,the returning step comprises redirecting the used cell culture mediumfrom a waste reservoir into the cell culture chamber. In otherembodiments, the method also involves discarding the used cell culturemedium if the parameter does not meet the predetermined threshold, andflowing fresh cell culture medium into the cell culture chamber.

In another aspect, this disclosure provides a cell culture system thatincludes a plurality of shelves for receiving fluid reservoirs. Theshelves may be stacked with a first shelf on top of a second shelf, eachof the first and second shelves configured to receive a fluid reservoir.Each of the shelves may include a retaining mechanism that retains thefluid reservoir on each of the first and second shelves.

The system may further include at least one pump, a processor operablycoupled to the at least one pump; and a substrate sized and configuredto hold a plurality of cell culture chambers at a same time. Preferably,the system further includes at least one sensor, and the processor maybe connected to the at least one sensor and can configured to operatethe at least one pump based on a characteristic measured by the sensor.For example, the sensor may measure a concentration of one or morecompounds within cell culture medium, such as glucose, lactate, or cellmetabolites. The processor may regulate the pump (e.g., turn the pump onor off) based on measurements made from one or more sensors. Forexample, one sensor may be attached to a cell culture chamber. Thatsensor may measure, for example, glucose levels inside the media withinthe cell culture chamber. When the glucose levels fall below apre-determined threshold, the processor may trigger the pump to replacethe media in the cell culture chamber.

In some embodiments, the processor is configured to receive and executeinstructions for culturing a cell type. In other instances, theprocessor may be configured to receive and execute instructions fortransducing T cells.

In some embodiments, the substrate is configured to receive cell culturechambers of different sizes and/or shapes. This configuration isadvantageous because it allows the system to be customized to culturedifferent quantities of cells, or different cell types, depending on theparticular needs of the user. The system may further include a pluralityof pumps. For example, the system may include a separate pump for eachcell culture chamber included within the system allowing for cellswithin each cell culture chamber to be separately cultured.

In some embodiments, the system further includes a plurality of tubesthat fluidically connect from a first fluid reservoir on the first shelfto the plurality of pumps, from the plurality of pumps to a plurality ofcell culture chambers, and from the plurality of cell culture chambersto a second fluid reservoir on the second shelf. Preferably, the systemis dimensioned for insertion into an incubator.

In a related aspect, this disclosure provides a method for the sterileculture of cells. The method includes providing a cell culture systemcomprising a plurality of shelves stacked with a first shelf on top of asecond shelf, each of the first and second shelves configured to receivea fluid reservoir; at least one pump; a processor operably coupled tothe at least one pump; and

a substrate sized and configured to hold a plurality of cell culturechambers at a same time. The method further includes loading a firstfluid reservoir onto the first shelf, loading a second fluid reservoironto the second shelf, loading a first cell culture chamber and a secondcell culture chamber onto the substrate, connecting the fluid and secondfluid reservoirs, the at least one pump, and the first and second cellculture chambers, with tubing; and operating the system to culture cellsinside the first and second cell culture chambers.

In some embodiments, the first and the second cell culture chambersincludes different sizes or shapes. In some embodiments, the systemincludes at least one sensor. In some embodiments, the processor isconnected to at least one sensor and is configured to operate the pumpbased on a characteristic measured by the sensor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an example of a multi-bioreactor system.

FIG. 2 shows an embodiment of a biological reactor.

FIG. 3 shows a multi-bioreactor system.

FIG. 4 shows a CAR-T/TCR workflow.

FIGS. 5-8 show comparisons of T cell expansion using different systems.

FIGS. 9-10 show a method for co-culturing freshly cultured dendriticcells and PBMCs or T cells, and results thereof.

FIG. 11 shows a method for forming cell-based immunotherapeuticproducts.

FIG. 12 shows a system architecture according to some embodiments.

DETAILED DESCRIPTION

The cell culture systems of the present invention significantly improveimmunotherapeutic product manufacturing, providing flow-basedimmunotherapeutic production technology with an unparalleled degree ofconsistency, quality, safety, economy, scalability, flexibility, andportability. In general, cells are grown in single-use cell culturechambers, sometimes referred to as cartridges, which are perfused at lowflow rates to achieve high expansion without the need for filters. Thesystem supports one or more cell culture chambers to be fluidicallycoupled to one another for carrying out the processing of a patient'scellular material to generate an immunotherapeutic product, as describedherein. It is to be understood that the bioreactors are provided in aclosed environment in certain embodiments. Scale-up of this exampleembodiment will be within the knowledge of the skilled artisan by addingmodules (e.g., biological reactors) to allow for serial and/or parallelprocessing. The skilled artisan will also appreciate that different oralternative arrangements may be desired based on the product to beproduced.

FIG. 1 shows an example of a multi-bioreactor system 900. The system 900includes a first cell culture chamber 820 and a second cell culturechamber 920, which have inlets 845 and 945 connected to tubing 940 influid communication with a fluid reservoir 980. The cell culturechambers have outlets 835 and 935 in fluid communication with wastereservoir 984. Pumps 910 a and 910 b facilitation pumping of fluid fromthe fluid reservoir 980 to the cell culture chambers 820 and 920. Thepumps are controlled by processor 999 in order to perform the functionsdescribed herein.

Another embodiment of a biological reactor 110 is shown in FIG. 2 ,which provides a more detailed schematic view of the parts of the cellculture chamber 120. It is important to note that the cell cultureplatform described herein is configured to allow cell culture chambersof different volumes, shapes, and physical characteristics to be used.The chamber shown in FIG. 2 is exemplary only, and other embodimentswill be apparent to the skilled artisan. As shown in FIG. 2 , the cellculture chamber 120 includes a bottom surface 122 and at least oneadditional surface 124. The bottom surface 122 is comprised of a firstmaterial to which cells adhere. In some embodiments the at least oneadditional surface 124 is comprised of a second material that gaspermeable. In other embodiments, which will be described in greaterdetail below, the entire cell culture chamber 120, including the surface124, is made of the first material which gas-impermeable. The cellculture chamber also comprises one or more inlets 126, 136 and one ormore outlets 128, 138. In certain embodiments, the biological reactoralso includes at least one perfusion fluid reservoir 132, at least onewaste fluid reservoir 134, at least one pump 140 for moving perfusionfluid through the chamber 120, as well as associated inlets 136 andoutlets 138 for transporting fluid to and from the reservoirs 132, 134and through the chamber 120.

With respect to the cell culture chamber 120, the first material can beany material which is biocompatible and to which antigen-presentingcells (APCs) or their precursors, such as dendritic cells (DCs) ormonocytes, respectively, will adhere. During the T-cell stimulation andexpansion process that occurs in the cell culture chamber 120, matureAPCs will develop and preferably adhere to the bottom surface 122,whereas the T cells remain in the supernatant above the bottom surface,making it easier to separately obtain the expanded T cells.

In one example embodiment, the first material comprises polystyrene. Onebenefit of using polystyrene for the bottom surface where culturing willoccur is a useful role that this material plays in the process ofgenerating dendritic cells from PBMCs. Specifically, polystyrenesurfaces can be used to enrich monocytes from a heterogeneous suspensionof PBMCs. This is a first step in the culture process utilized togenerate DCs by differentiation of monocytes via culture in mediumcontaining, for example, IL4 and GM-CSF. The use of the same polystyrenesurface for dendritic cell production all the way through one cycle of Tcell stimulation is tremendously valuable from a bioprocess standpointas it eliminates a large number of transfer steps that would otherwisebe necessary, thereby allowing for a closed system for DC-stimulatedtherapeutic T cell manufacturing.

The bottom surface can have a surface area comparable to conventionalwell plates, such as 6- and 24-well plates (9.5 cm² and 3.8 cm²,respectively). It is also to be understood that the surface area can besmaller or even much larger than conventional well plates (e.g., havingsurface areas comparable to standard cell culture dishes and flasks),such as having a surface area between about 2.0 cm² and about 200 cm²,for example, about 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0,12.0, 13.0, 14.0, 15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 25.0, 30.0, 35.0,40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 100.0, 125.0, 150.0,175.0, and 200.0 cm², and any surface area in between.

The at least one additional surface 124 can comprise any configuration,such as one or more side walls and a top wall. In one embodiment, asshown in FIG. 2 , the side walls can be arranged at 90 degree angleswith respect to one another, such that a box shape is formed inconjunction with the bottom surface 122. In another embodiment, the atleast one additional surface 124 forms a curved side wall, such that thechamber 120 or a cross-section thereof forms a cylinder, ellipticcylinder, cone, dome-like shape, or triangular shape. It is to beunderstood that the above example configurations are non-limiting andthat the at least one additional surface can have other configurationsnot provided in the aforementioned example configurations.

An example configuration of a multi-bioreactor system can be found inFIG. 3 , with additional detail regarding the processes carried outusing this configuration provided below. As shown in FIG. 3 , panel B,in the event that a second bioreactor 210 is involved, the second cellculture chamber 220 is positioned to connect with the first cell culture120 chamber via the outlet of the first chamber and the inlet of thesecond chamber. The connection is preferably a sterile connection. Theconnection allows for the injection of sterile air into the first cellculture chamber 120 to transfer the supernatant containing the expandedT cells into the second cell culture chamber 220. Alternative techniquesknown in the art of fluid flow may be employed to transfer thesupernatant from the first cell culture chamber 120 to the second cellculture chamber 220. As also shown, each bioreactor includes its ownfluid and waste collection reservoirs, pumps, and associated tubing.However, it is to be understood that the reservoirs and pumps can beshared between bioreactors.

The system is configured to be able to perfuse the cells in the cellculture chamber with medium, which is required for various methods ofcell culture described herein. Perfusion ensures uniform nutrient andcytokine supply to the cellular mixture along with sufficient gasexchange and waste removal to assist with the formation of thecell-based immunotherapeutic product. Maintaining consistent localconcentration profile of medium and cytokines ensures greater yields andthe potential ability to speed up the process of monocytedifferentiation to DCs compared to prior art plate-based protocols.However, the combination of adherent (DC) and non-adherent (T cell)types, along with the high sensitivity of DCs to mechanical forces poseschallenges to the stimulation and expansion of antigen-specific T cells,especially with respect to the flow of fluid through the cell culturechamber. Thus, in those embodiments in which medium and cytokines areprovided via perfusion, systems of the present invention must be able tosupply cells with nutrients and cytokines without removing cells fromthe bioreactor while also taking into account the shear sensitivity ofcertain antigen-presenting cells, such as DCs. Systems and methods ofthe invention aim to optimize retention of autocrine/paracrine signalsfavoring T cell proliferation while refreshing growth factors andmaintaining minimal physical stimulation of DCs. In order to account forthis, both the direction and the rate of perfusion flow through the cellculture chamber must be taken into consideration.

In certain aspects, the fluid flow rate is maintained below thesedimentation rate of the antigen-presenting cells. As such, theantigen-presenting cells will remain within the culture chamber becauseof their mass. In other words, the antigen-presenting cells will sinktoward the bottom of the cell culture chamber and therefore remain inthe cell culture chamber.

A flow rate that is lower than the sedimentation rate can be calculatedaccording to Equation 1:

v_max=[((ψd_p))]{circumflex over ( )}2/150μ,g(p_cell−p_liquid)e{circumflex over ( )}3/(1−e)

where v_max is the liquid velocity beyond which cells will be liftedupwards, ψ is shape factor of cells (ratio of surface area of the cellsto surface area of a sphere of equal volume; note that cells are notperfectly spherical and this factor is expected to be below 1), d_p is adiameter of a spherical particle of volume equal to that of a cell, μ isviscosity of liquid containing cells, g is the gravitational constantp_cell is the density of cells, p_liquid is a density of liquidcontaining cells, and e is a fraction of the volume of interest that isnot occupied by cells.

In the methods described below that involve perfusion of medium, itshould be understood that perfusion may be performed continuously duringculturing or it can occur at specific points in time over the timeperiod in which the cells are cultured in any one cell culture chamber,such as, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times eachday or week. Continuous perfusion helps to maintain a near constantculture volume throughout the process. Likewise, cytokines can beinfused at one or more points during culturing, such as, for example, 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more times, or continuously. In thoseembodiments, the continuous perfusion helps maintain a consistent localconcentration profile of cytokines, which can help to ensure greateryields and has the ability to increase the speed at which T cells arestimulated and expanded compared to static cell culture methods.

Perfusion parameters can be varied at any time during a culture cycle.Example parameters include, but are not limited to, the median flowrate, cytokine concentration, and duration of culture cycle. Each ofthese parameters may have an impact on the efficacy of T-stimulation.For example, in recent work designing culture chambers formonocyte-diffusion to DCs, as described in International PatentApplication Nos. PCT/US2016/040042 and PCT/US2016/60701, it has beendetermined that medium perfusion rates corresponding to wall shearstress levels of 0.1 dyn/cm2 are capable of producing DCs that arephenotypically identical to those generated using conventional 6- or24-well plate-based protocols. As such, by measuring the one or more ofthe phenotypic and functional measures described above during theculture cycle, the effect of one or more perfusion parameters onefficacy can be monitored, allowing for appropriate adjustments.

To facilitate perfusion, the system includes one or more pumps 140. Thepumps can be operably coupled to the cell culture chamber 120 forperfusing perfusion medium into the cell culture chamber. Thebioreactors 110 can also include one or more fluid reservoirs 132. Thefluid reservoirs 132 are in fluidic communication with the cell culturechamber 110 and can be operably coupled to one or more pumps 140. One ormore tubes for connecting the fluid reservoirs to the pumps and cellculture chamber are also provided. In certain aspects, the one or morepumps are configured for pumping fluid from the fluid reservoir, throughthe cell culture chamber, and into the waste collection reservoir. Inthe example embodiment shown in FIG. 2 , fluid moves from the fluidreservoir 132, through tubing 152 to the pump 140 and into the cellculture chamber 120 via inlet 136, back out of the cell culture chamber120 via outlet 138, through tubing 154, and into the waste collectionreservoir 134.

In certain embodiments, the fluid reservoir and/or waste collectionreservoir can each be provided as one or more sealed bags or containersfluidically coupled to the chamber. Each reservoir contains an inletport and an outlet port, or an outlet port and a vent fluidicallycoupled to the inlet of one or more cell culture chambers. In someembodiments, Luer connectors and silicone gaskets cut to fit around theLuer connectors can be used to prevent leakage through either or both ofthe inlet or outlet. In some embodiments, the sealed reservoirs can beconnected to the cell culture chamber using a sterile tube weldingdevice that creates a fluidic connection without exposing either vesselto the outside environment and maintaining sterility. As will bediscussed in greater detail below, this allows methods of the inventionto be performed in a closed system.

Due to the small size and portability of the disclosed cell culturesystems, they can be easily used in conjunction with a tube weldingdevice. Systems of the invention can be easily lifted and carried intoproximity with a tube welder in order to make the necessary sterileconnections. The size and configuration of the cell culture systems alsomakes them compatible with standard incubators. The cell culture systemsare sized and configured to fit on a single shelf inside a conventionalincubator, such that the disclosed processes can be carried out therein.Multiple instruments can fit in a single incubator, depending on theconfiguration. Conditions within the incubator include sustainedtemperatures of 37° C. and 95-100% humidity. Thus, the materials chosenmust have the integrity to withstand these conditions, given that thematerials (including fluids and biologics) tend to expand under suchconditions. Furthermore, in some circumstances, conditions within theincubator remain stable, and automated recording of the temperature ispossible to have knowledge of temperature fluctuations to correlate withany aberrations in the reactions performed in the incubator.

Accordingly, any supply of power should not change the environmentwithin the incubator. For example, certain pumps generate heat.Accordingly, in one embodiment, the pumps are housed separately from thebiological reactors, but are still in fluidic and operablecommunications with the reactors. In another embodiment, the pumps aredirectly attached to the biological reactors and located within theincubator, but are heat free or are operably connected to a heat sinkand/or a fan to dissipate the heat. In another embodiment, the pumps runon a duty cycle to reduce the amount of heat generated. Regardless ofthe configuration, the pumps are operably coupled to the biologicalreactors, and, in turn, the cell culture chambers. In some embodimentsthe system also includes a heater for controlling the temperature of thecell culture reservoir and optionally the fluid reservoir. In such aconfiguration, no incubator is required, and the system can operateautonomously, with only a source of electrical power. If the systemlacks a heater, it can be operated inside of a cell culture incubator.

Additional details regarding perfusion-based automated cell culturesystems, such as small scale culture system for endothelial cell culturewith on-board reagent storage and perfusion enabled by an on-boarddisposable peristaltic pump and a larger scale culture system fordendritic cell generation from monocytes using chambers with polystyrenebottom surfaces, can be found in international patent publications WO2017/004169; WO 2017/079674; and WO 2018/005521; as well as U.S. patentapplication Ser. No. 16/539,916; each of which is incorporated herein byreference in their entirety.

Systems, or devices, of the invention are modular and capable of fluidicconnection to other similar devices in series (i.e., with fluid flowingfrom one device into another) and/or in parallel, and may also be soconfigured as to physically stack with one another or be capable ofphysical arrangement within a related device such as an incubator. Themodular design of the system specifically allows for modules to beflexibly switched in and out depending on a desired process to beincluded within the system.

Fluidic devices of the invention, including the biological reactorscomprising cell culture chambers, can be provided in either amicrofluidic embodiment (i.e., wherein one or more channels or chamberstherein has a dimension in the range of from about 1 μm to about 999 μm)or a macrofluidic embodiment (wherein all of the channels or chamberstherein have dimensions of about 1 mm or more), or both.

The fluidic devices can further include additional fluid channels orcompartments, gaskets or seals, mixing zones, valves, pumps, vents,channels for pressurized gas, electrical conductors, reagents, ports,and tubing as required by a particular design. They also may contain oneor more control modules, transmitters, receivers, processors, memorychips, batteries, displays, buttons, controls, motors, pneumaticactuators, antennas, electrical connectors, and the like. The devicespreferably contain only materials that are nontoxic to mammalian cellsand that are compatible with sterilization by the use of alcohol and/orheat or other means, such as exposure to gamma radiation or ethyleneoxide gas.

The materials of equipment are chosen with the appropriate chemicalcompatibility under different temperature and pressure rating specificto each process. Additionally, the choice of pumps implemented in thedevice, such as syringe, peristaltic, pressure, and rotary pump, rangesfrom a nL to a mL in flow rates and 10 to 10,000 psi in pressuredepending on the flow and pressure requirements for the differentfunctions.

Systems of the invention can also include one or more sample solutionreservoirs or well or other apparatus for introducing a sample to thedevice, at various inlets of the modules, which are in fluidcommunication with an inlet channel. Reservoirs and wells used forloading one or more samples onto the fluidic device of the presentinvention includes but are not limited to, syringes, cartridges, vials,Eppendorf tubes and cell culture materials (e.g., 96 well plates).

Where useful, surfaces of the devices can be made more hydrophilic, suchas by exposure to a plasma, or can be coated with one or more gels,chemical functionalization coatings, proteins, antibodies,proteoglycans, glycosaminoglycans, cytokines, or cells. Fluidic devicesof the invention are preferably devoid of fluid leaks under operatingconditions and capable of sterile operation over a period of days toweeks. Fluidic devices of the invention also include a samplingmechanism that allows fluid to be removed from the system for testingwithout introducing new material or contaminants to the system.

In certain aspects, at least part of the cell culture system comprisesdisposable components, some or all of which can be housed within anon-disposable frame. In other aspects, all components of the system aredisposable. Furthermore, in some embodiments, the cell culture systemincludes a sample tracking component for tracking and documentingpatient material.

At least one step, and sometimes a plurality or all steps, during themanufacturing process are monitored for product characteristics (e.g.,purity and polymorphic forms) using a variety of inline processanalytical tools (PAT) or miniaturized micro-total analysis system(micro-TAS).

As described above, the cell culture systems of the present inventionare capable of controlling the direction and flow of fluids and entitieswithin the system. Systems of the invention can use pressure drive flowcontrol, e.g., utilizing valves and pumps, to manipulate the flow ofcells, reagents, etc. in one or more directions and/or into one or morechannels of a fluidic device. However, other methods may also be used,alone or in combination with pumps and valves, such as electro-osmoticflow control, electrophoresis and dielectrophoresis (Fulwyer, Science156, 910 (1974); Li and Harrison, Analytical Chemistry 69, 1564 (1997);Fiedler, et al. Analytical Chemistry 70, 1909-1915 (1998); U.S. Pat. No.5,656,155).

Systems of the invention can also include or be operably coupled to oneor more control systems for controlling the movement of fluid throughthe system; monitoring and controlling various parameters, such astemperature, within the systems; as well as detecting the presence ofcell-based immunotherapeutic products, quantity of product (directly orindirectly), conversion rate, etc. The system may also be equipped withnumerous classes of software, such as an advanced real-time processmonitoring and control process, allowing for feedback control, as wellas processes that allow integration and scale-up given reaction andpurification results obtained using the system.

In certain embodiments, the system includes a combination of micro-, ormacro-fluidic modules and tubing that are interchangeable in terms ofchannel dimensions, flow geometry, and inter-connections between thedifferent modules of the device. Each module and tubing may be designedfor a specific function. In one embodiment, all of the modules withinthe system are designed for cell culturing and T-cell stimulation. Inother embodiments, the modules with the system are designed fordifferent functions, such as tissue processing, dendritic cellgeneration, cell culturing, concentration, and/or purification, allintegrated for the continuous manufacturing of an immunotherapeuticproduct. Both homogenous and heterogeneous processes are consideredwhich are suitable for flow application. These processes are designedand optimized with respect to the starting materials and operatingconditions, such as temperature, pressure and flow rates so as to notreadily clog the system during the flow process.

Gas-Impermeable Cell Culture Chambers

In some embodiments, the cell culture chambers of the disclosed systemare made of a gas-impermeable material. The gas-impermeable material isbiocompatible and is a material to which dendritic cells will adhere. Inone example embodiment, the gas-impermeable material comprisespolystyrene, which as described above is useful for enriching monocytesfrom a heterogeneous suspension of PBMCs. The entire cell culturechamber being made of the gas-impermeable material offers a largersurface area to which cells can adhere and increases the sterility ofthe system.

A gas-impermeable cell culture chamber can be substantially the same asthe chamber 120 shown in FIG. 2 and can have any volume, shape, size,and physical characteristic described above, with the exception that theadditional surface 124 and all other surfaces of the chamber 120 aremade of the same material as the bottom surface 122. In someembodiments, the top, bottom, and all side walls of the chamber 120 aregas-impermeable. The bottom surface of the gas-impermeable cell culturechamber can have a surface area comparable to conventional well plates,such as 6- and 24-well plates (9.5 cm² and 3.8 cm², respectively). It isalso to be understood that the surface area can be smaller or even muchlarger than conventional well plates (e.g., having surface areascomparable to standard cell culture dishes and flasks), such as having asurface area between about 2.0 cm² and about 200 cm², for example, about2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0,15.0, 16.0, 17.0, 18.0, 19.0, 20.0, 25.0, 30.0, 35.0, 40.0, 45.0, 50.0,55.0, 60.0, 65.0, 70.0, 75.0, 100.0, 125.0, 150.0, 175.0, and 200.0 cm²,and any surface area in between.

Certain modifications need to be made to the system described above whenthe chamber is not permeable to gas. For example, in such embodimentswhere gas does not flow through one of the surfaces of the cell culturechamber, the gas exchange between cells and the medium must befacilitated in another way. The cell culture chamber comprises one ormore inlets 126 and 136 and one or more outlets 128 and 138. The inletand outlet openings can be fluidically coupled to tubing, which issealed with the respective opening. The inlets and outlets are therebyconnectable to a perfusion fluid reservoir and a waste fluid reservoirwith corresponding pumps for moving perfusion fluid through the chamber.The inlets and outlets can be located in any surface of the chamber. Insome embodiments, they are located in the top of the chamber. The tubingis preferably high-permeability tubing, In order to effectuate gasexchange, the medium can exchange gas prior to entry into the chamberand after leaving the chamber through the high-permeability tubing. Gasis therefore effectively brought into the chamber through the one ormore inlets and removed through the one or more outlets. The inlets orthe tubes connected thereto can include a filter such as a 0.2 micronfilter, for filtering liquid or air entering the cell culture chamber.

Gas flow is affected by perfusion rates, the parameters of which can becontrolled as described above. By exchanging gas via the highpermeability tubing, the system maintains the ability to achieve therequired levels of gas exchange without requiring the chamber to be gaspermeable. Methods of cell culture can be performed in a completelygas-impermeable chamber, with inlets 126 and 136 and outlets 128 and 138for perfusion and gas flow. In methods embodiments, the gas-impermeablecell culture chambers can be used to culture cells by loading cells intothe chamber and perfusing them by flowing cell culture medium in and outof the chamber via the inlets and outlets. The perfusion flow providesnutrients as well as gas exchange to the cell culture. Because flowthrough the chamber is laminar, some methods may require additionalshaking, such as for cell harvesting. However, given the size andconfiguration of the disclosed systems, the entire system can be placedon an orbital shaker as needed.

Another advantage of gas-impermeable embodiments is that they are easierto manufacture because they have fewer different parts and materials.They can be made as large or small as needed. The gas impermeablechamber can be integrally formed or it can be formed from multipleparts. For example, it can be formed out of a single piece of materialby traditional manufacturing processes or additive manufacturingprocesses such as 3D printing. In embodiments, a plurality of members,each made of the gas-impermeable material, are joined together usingmethods known in the art, such as mechanical fastening, adhesive andsolvent bonding, and welding, such as ultrasonic welding.

Interchangeability of Cell Culture Chambers

Cell culture systems of the present invention are configured to be ableto connect with cell culture chambers of various sizes and shapes. Thecell culture system can include a fluid reservoir, a waste reservoir,and one or more pumps for controlling fluid flow to and from thereservoirs. The cell culture system also has an area configured toreceive one or more cell culture chambers of different shapes and sizes.One or more tubes fluidically connect the fluid reservoir, the cellculture chambers, and the waste reservoir. The pumps are configured tomove fluid through the tubes.

Different cell culture chamber sizes can be used for different purposes,or in combination with each other. The size can be selected based on thedesired cell output, or different proportions of reagents needed. Forexample, small cartridge sizes can be useful for research, pre-clinicaluses, or process development. Large cartridges are a higher capacityversion with the same architecture.

The height of the one or more cell culture chambers can vary. Forexample, and not limitation, an example range of cell culture chamberheights includes heights of anywhere from 0.5 mm to 100 mm, such as 0.5,1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 15.0, 20.0, 25.0,30.0, 35.0, 40.0, 45.0, 50.0, 55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0,90.0, 95.0, 100.0 mm, or more, or any height therebetween. In certainembodiments, the heights of the chamber can be comparable to liquidheights in cultures that are typically performed in 6- and 24-wellplates, such as between 2 and 6 mm, with a volume capacity of about 0.8mL to 6 mL. In other embodiments, the cell culture chambers will be oflarger size, such as between 10 mm and 50 mm, with a culture surface ofabout 50 cm². In some embodiments the cell culture chamber has a volumecapacity of between about 1 mL and about 100 mL, and may beapproximately 5, 10, 20, 25, 30, 40, 50, 60, 70, 80, or 90 mL, oranywhere in between. In other embodiments the cell culture chamber has avolume capacity of between about 100 mL and about 1,000 mL, for example100, 200, 300, 400, 500, 600, 700, 800, 900, or 1,000 mL. In aparticular embodiment a cell culture chamber has a capacity of 210 mL.

The interchangeability of cartridges with the present invention allowsscaling up during a cell growth procedure using the same system. Thisavoids the need to switch to a different cell culture system in order tocontinue growing cells. For example, to manufacture a batch size ofabout 2 billion cells, the system can start with a small cartridge witha capacity of about 25 mL, and then scale up to a large cartridge with acapacity of about 210 mL. This interchangeability means that more partsof the process can be done on the same system, from activation throughfill and finish. Depending on the needs of a particular protocol, thesystem is operable with, for example, two large (approximately 210 mL)cell culture chambers, two small (approximately 25 mL) cell culturechambers, or one of each. Since each inlet and outlet can be connectedto any size chamber, the system is capable of scaling up or scaling downas needed.

With reference once again to FIG. 1 , the system 900 includes a platform950 configured to support one or more cell culture chambers 820 and 920.Regardless of shape or size, the cell culture chambers can be connectedvia tubes 940. In this way, cell culture chambers of different shapesand sizes are compatible with the system. The cell culture chambers canbe arranged in different configurations on the platform, such aspositioned side-by-side or stacked. In embodiments, the tubes 940 areintegrally formed with the chamber. The tube and the chamber body can bejoined together using the same manufacturing methods discussed above. Inone embodiment, the cell culture chamber is manufactured with at least aportion of the material in the side and/or top walls cut out to allowfor the formation of the one or more inlets or outlets. In an examplearrangement, a tube can be separately inserted into the openings to forma seal with the vessel. It is to be understood that the aforementionedconfigurations are only examples and that other configurations forjoining the chamber and one or more tubes are also contemplatedembodiments of the present invention.

Interchangeability is facilitated in part by using sterile tubeconnections to couple the various vessels and chambers of the system.The sterile tubes are preferably connected using a sterile tube welder.Generally a sterile tube welder is a device that can receive two tubes,secure them in place, and then cut them with a hot blade, realigns themso that the first tube and second tube are aligned, and melts the twocut ends together when the blade is retracted. Sterile tube welders foruse with the present invention can be any commercially available steriletube welder, including SCD® IIB from Terumo BCT, Inc. (Lakewood, Colo.);the Vante® 3690 from Vante BioPharm (Tucson, Ariz.); and the TCD® fromGenesis BPS™ (Ramsey, N.J.).

As will be described in greater detail below, in certain embodiments thesystem is functionally closed. The closed system is maintained by all ofthe transfers being done using sterile tube welding. For example, cellsmay be stored in a sterile bag, which is then connected to a sterilecell culture chamber. The cells can be flowed into the cell culturechamber while maintaining sterility. The tube connectivity allows cellseeding, washing, and harvesting to all be done on the same device understerile conditions. By using the sterile tube welder, one bag does nothave to be disconnected before connecting another.

The above description focuses on the system components and variouspossible configurations. The following description focuses on certainprocesses that can be carried out using systems of the invention.

CAR-T and TCR Processes in a Closed System

The cell culture systems described above are useful for methods ofproducing CAR-T and TCR transduced T cells. A visualization of aCAR-T/TCR workflow is shown in FIG. 4 . The disclosed system can be usedto perform several of the steps of the workflow in a closed system,including genetic modification, cell expansion, cell washing andconcentration, and cell formulation. Prior known methods used in theindustry producing CAR-T and TCR transduced T cells are not performed ina closed system. Commonly used polystyrene T flasks have to be openedand closed to perform transfers, and are therefore subject to potentialcontamination. The present invention uses closed and interchangeablecartridges made of solid polystyrene, which allows protocols designedfor T flasks to be easily reformatted to be done on the disclosedsterile system.

In some embodiments, a method of the invention involves flowing cellculture medium into a culture chamber with T cells, and perfusing the Tcells to transduce them with a transduction reagent such as an inactivevirus expressing CAR or TCR, for example. The perfusion fluid mayinclude an activation reagent to expand the cells. In some embodimentsthe transduction and/or activation reagents are premixed with the cells,and in other embodiments they are from a separate sterile bag or vessel.The bags can be connected through sterile welding means as describedabove, and the reagents can be flowed into the bag by gravity or by apump. In some embodiments, the cell culture chambers are filledcompletely with little or no headspace.

The cells are grown for up to about 3 days, during which time they areallowed to take on CAR, TCR, and are sustained by the perfusion medium.The cells form a sediment in the chamber and the perfusion rate ismaintained low enough to prevent the cells from flowing out of thecartridge. The transduced cells are expanded in the culture chamber andthen can be transferred to a larger culture chamber, which is connectedvia a sterile tube, for further expansion. Using methods of theinvention a 7-day expansion in a small cartridge (25-mL volume) canyield about 500 million to one billion T cells, and a large cartridge(210-mL volume) can yield about one to three billion T cells.

The connection with the perfusion bag is detached and a harvest bag isattached. The cells are then drained into the harvest bag. Inembodiments, a buffer bag can be connected by the same methods and usedto perform one or more washes of the cells before they are removed intothe harvest bag. The volume of liquid that the cells are in can beincreased or decreased, by draining one liquid, adding another, andresuspending the cells in the new volume of liquid. In some embodiments,the system can be drained to remove built-up lactic acid. As the cellculture expands, lactic acids builds up and may not be removed fastenough through perfusion alone. A solution is to drain the medium toreduce the total volume (by perhaps 90-95%) without removing the cells,and then perfuse with fresh medium. In some embodiments, the medium canbe replaced with a different cell culture medium or cryopreservationmedium.

The cell culture chambers are connected in a closed system such that theentire method is performed in a closed environment without the need toexpose the media to air by opening any of the vessels when transferring.As has been mentioned, all of the connections and disconnections of themethod can be done with a sterile tube welder.

Unlike the prior art, the disclosed method of activation, transduction,and expansion are performed in a closed sterile system. With theinterchangeability described above, a user can scale up to a batch of upto 10-20 billion expanded cells, all in a closed environment. In someembodiments, the methods may be used to prepare a batch formanufacturing on a larger bioreactor system. In conventional CAR-Tmanufacturing, batches of 10 billion or more cells are needed. Thepresently disclosed systems and methods can perform the upstream stepsof activation, transduction, and initial expansion, prior totransferring 1 billion or more cells to a larger expansion system, suchas XURI™ available from GE Healthcare (Chicago, Ill.). This capabilitymakes the systems highly compatible with non-magnetic activationreagents.

FIGS. 5-6 show an example of the comparison between a T cell expansionconducted with the presently disclosed system, known as BATON™ fromFlaskworks, LLC (Boston, Mass.), and another commercially available Tcell expansion platform G-REX® from WilsonWolf (St. Paul, Minn.). FIG. 5shows a graph of the fold-expansion using BATON™ over 9 days with PBMCsstimulated with DYNABEADS® from Thermo Fisher (Waltham, Mass.) run in a25 mL cartridge. The robust fold-expansion achieved with BATON™ iscomparable to that of G-REX®.

FIG. 6 shows the number of cells on day 0 and day 7, using both systems.With BATON™, approximately a billion cells were obtained from 40 millionT cells in seven days in a 210 mL cartridge in accordance with thepresent disclosure. The phenotype is predominantly central memory. Asfurther shown in FIG. 7 , phenotypic profiles are comparable betweenBATON™ and G-REX®. The figure also compares T75 flasks available fromCorning Inc. (Corning, N.Y.). As shown, both BATON™ and G-REX® generatedequivalent CD4/CD8 ratios for Day 9.

As shown in FIG. 8 , the cytotoxicity of T cells produced with BATON™ iscomparable to that of G-REX®. The effector cells were expanded for 9days, and the target cells were Jurkat T cells. Cells were mixed at a10:1 ratio of effector-to-target and incubated at 37° C. at 5% CO₀ for24 hours. The medium was RPMI 1640 (ATCC)+15% FBS. The Day 5 and Day 7cells from all three groups were comparable in cytotoxicity.

Neoantigen Process on a Closed System

The disclosed systems are also useful in a workflow for producingneoantigen-targeting T cells. This class of therapeutics involves theco-culture of antigen-presenting cells stimulated with libraries oftumor-specific peptides and autologous T cells. Manufacturing ofneoantigen presenting cells is more complex than CAR-T. Unlike CAR/TCR,the present method can pursue a library of targets rather than just one.It requires certain capabilities provided by the presently disclosedsystems which are not available from prior art systems.

The present system generates fresh dendritic cells from patientmonocytes. The system also is configured to co-culture dendritic cellswith patient PBMCs or T cells and deliver stimulated T cells. The systemcan perform multiple cycles of co-culture with freshly generateddendritic cells to avoid competing effects between different antigens.Parallel processing of dendritic cells and T cells to facilitate thisprocess will be described in greater detail below. The typical dosesizes for neoantigen therapies are about 200 million T cells, which canbe handled by the disclosed system in either a small (approximately 25mL) or large (approximately 210 mL) cartridge.

Unlike prior art methods, systems of the invention facilitate all of theabove in a closed environment. The method utilizes the interconnectedcell culture chambers of the present system. Purified monocytes areintroduced to one of the cell culture chambers, and purified T cells areintroduced to the other. The monocytes are perfused with cell culturemedium to produce dendritic cells, which are then contacted with antigenmaterial comprising tumor-specific peptides. This produces maturedendritic cells presenting tumor-specific peptides. T cells aretransferred into to chamber containing the mature dendritic cells toco-culture them with the T cells. Co-culturing producesneoantigen-targeting T cells. After one cycle of stimulation viaco-culture, the T cells can be removed and optionally flowed into asecond chamber containing freshly generated mature dendritic cells toperform a second co-culture. The second batch of dendritic cells can beproduced asynchronously from the dendritic cells generated in the firstchamber.

In embodiments, mature dendritic cells are generated in a first chamberand then T cells added to the first chamber and co-cultured. T cellsfrom the first chamber are then moved to a second chamber where they areco-cultured with freshly generated dendritic cells that are stimulatedwith either the same or a different set of peptides. The expanded Tcells can then be moved back to the first chamber where yet anotherbatch of fresh mature dendritic cells stimulated with either the same ordifferent set of peptides await.

The T cells can be transferred back and for the between two connectedchambers any number of times as desired. This can be particularly usefulfor stimulating DCs with several different peptides. For example, if onehas a library of 20 or so peptides with which the DCs need to bestimulated, attempting to stimulation the DCs with all peptides at oncewould result in insufficient stimulation because of competing effectsbetween the peptides. Some peptides have greater effect and/or affinitythan others. Performing the stimulation in stages, for example,stimulating a batch of DCs with five peptides in a first co-culture, andthen stimulating the next batch of DCs with five different peptides in asecond co-culture, and so on. In other embodiments, multiple stimulationcycles can be done with a single, small group of peptides. Innonlimiting embodiments, the T cells can be transferred 2, 3, 4, 5, 6,7, 8, 9, 10, 15, 20, or 25 times. Each co-culture can involve DCsstimulated with the same or different peptides.

The entire method is performed within the closed system of the presentdisclosure, thereby maintaining sterility throughout the method. Likethe other disclosed methods, the culture chamber connections can beachieved with sterile tubes that are connected with a tube welder. Thismaintains that sterility of the cell culture medium that is provided ina sterile vessel. In various embodiments, the T cells can be transferredbetween chambers on the same instrument or transferred between chamberson separate instruments.

After the co-culture is complete, fluid is drained from the chamber, andthe neoantigen-targeting T cells can be washed with a buffer andresuspended in a cryopreservative and/or harvested in a harvest bag,which is also connected in a closed manner.

FIG. 9 shows a schematic flowchart of a method for co-culturing freshlycultured dendritic cells and PBMCs or T cells on the closed system ofthe present invention. Using sterile welded connections, the disclosedcell culture system provides automated seeding, culture, and cellharvesting. As shown in the workflow, monocytes are seeded at day 0, anddifferentiate into dendritic cells over days 0-6. At day 6, allogeneicPBMCs or T cells are added. Dendritic cells enable T cell expansion fromPBMCs. The expanded T cells are harvested on day 13.

Expanded T cells produced with the disclosed methods show a robustcytotoxic ability. Results of an example performed using the workflow isshown in FIG. 10 . The target ratio of CD 8+ T cells to Jurkat cells was1:1, with experiments performed using 1-3 million harvested cells.Jurkat cells were stained with PKH67 prior to the assay. Cells wereincubated for 1 day, and all cells were stained with CD3 and Annexin Vpost-assay. The resultant dead Jurkat cells are shown in FIG. 10 .

Parallel Processing of Dendritic Cells and T Cells

The process for forming cell-based immunotherapeutic product requiresco-culturing two types of cells. With the presently disclosed systems,these cells can be generated in parallel for more efficient generationof antigen-specific T cells. In brief, dendritic cells are produced frommonocytes and matured by contact with antigen material, and T cells areactivated and then co-cultured with the DCs. In prior art methods, thismethod would require several manual steps. The current system howevercan produce the two cell batches in parallel in a closed system. Usingthe systems disclosed herein, dendritic cells are produced in onechamber and, in parallel, T cells are stimulated in another connectedchamber. Monocytes are perfused in a first chamber to produce DCs, whichare contacted with antigen material to mature them. Activated T cellsfrom another connected chamber are flowed into the first chamber tocontact the DCs and further culture the T cells. Like in the othermethods, the chambers are connected via a sterile tube, so that themethod is performed in a substantially closed system. The T cells can becollected by flowing them into a collection vessel and/or transferred toa cryopreservation medium, while still in a closed system configuration.

Reference is made to FIG. 11 which shows a general overview of a processfor forming cell-based immunotherapeutic products. The steps ingenerating cellular therapeutic product in accordance with certainembodiments of the present invention include the co-culturing ofstimulated antigen-presenting cells (e.g. DCs) with T cell containingcells in a biological reactor containing a cell culturing chamber. Asupernatant containing expanded therapeutic T cell products is generatedduring culturing. In certain aspects, in order to produce a quantity ofantigen-specific T cells sufficient to elicit a therapeutic response ina patient, the T cells must undergo additional culturing in one or moreadditional cell culturing chambers. In order to effectuate thisadditional culturing, the transfer of supernatant from the culturechamber in which the supernatant was generated to a subsequent cellculture chamber containing a fresh supply of antigen-presenting cellsmust occur. The transfer of supernatant between cell culture chambersmay involve the introduction of a gas flow into the first cell culturechamber that transfers the supernatant comprising the first cell productthrough a fluidic connector and into the new cell culture chamber.Furthermore, during each of the culturing steps, perfusion fluidcontaining, for example, medium and cytokines, can be perfused to thechambers. In certain aspects, the perfusion fluid flows through thechambers along a vertical flow path so as to ensure that the cellsremain within the chamber during culturing. One or more subsequent cellculture chambers can be connected to the system with each chambercontaining a new batch of antigen peptide-pulsed autologousantigen-presenting cells.

In order to stimulate and expand antigen-specific T cells, the processbegins with a co-culture of T cell containing cells withantigen-presenting cells (APCs) obtained from the same individual in acell culture chamber. In a particular embodiment, the T cell containingcells include peripheral blood mononuclear cells (PBMCs) and the APCsinclude DCs. The T cell containing cells and APCs can be provided to thecell culture chamber in a ratio (T-cell containing cells:APCs) fromabout 1000:1 to 1:1000 of about, such as, for example and notlimitation, 1000:1, 900:1, 800:1, 700:1, 600:1, 500:1, 400:1, 300:1,200:1, 100:1, 75:1, 50:1, 25:1, 20:1, 15:1, 10:1, 5:1, 4:1, 3:1, 2:1,1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 1:15, 1:20, 1:50,1:75; 1:100, 1:200: 1:300, 1:400, 1:500, 1:600, 1:700, 1:800, 1:900,1:1000, or any ratio therebetween. In one aspect, a ratio of 10:1 ispreferred.

In order to initiate stimulation and expansion of T cells from theinteraction of APCs with T-cell containing cells, the APCs need to bestimulated. This can be done through the use of one or more stimulatorymolecules. In certain embodiments, the stimulatory molecule is non-tumorspecific. In other embodiments, the stimulatory molecule is tumorspecific. For example, the stimulatory molecule can be chosen from oneor more characteristics of an individual's tumor, such as differentantigen peptides. In some embodiments, the stimulatory molecule ispreferably added only in the beginning of a culturing cycle. Thestimulatory molecule can be added over a period of only about a fewminutes, an hour, a few hours, or longer. In one preferred embodiment,the stimulatory molecules are added over about an hour time period.

The co-culturing of APCs and T-cells takes place in a culture medium.Example culture media include, but are not limited to, RPMI medium, andCellgenix® medium. Any other suitable culture medium known in the artcan be used in accordance with embodiments of the present invention.Cytokines such as IL-4 and GM-CSF can also be added to the culturemedium.

It is not usually sufficient to do only one co-culture. The disclosedsystem allows T cells to be pulled out in a suspension. Here T cells canbe re-stimulated with fresh dendritic cells and multiple co-cultures canbe done in the closed system. With the parallel processing methods ofthe present invention, the cartridges can be connected in a chain andcells can be pushed from one cartridge to another. One reactorcontaining mature, adhered DCs will be loaded with PBMCs and subjectedto a stimulation cycle with perfusion of medium and cytokines. Withparallel processing, the expanded T-cells can be continuously exposed tofresh DCs being produced in the first cell culture chamber by beingpulsed with antigen peptides. The stimulation process can continue foras long as needed in order to generate a sufficiently large number ofcells for a therapeutic dose of T cells.

Connecting multiple chambers together via a sterile connection caninvolve the configuration of multiple chambers as discussed above withrespect to FIG. 3 . The connection allows for the injection of sterileair into the first cell culture chamber to transfer the supernatantcontaining the expanded T-cells into the second cell culture chamber. Incertain embodiments, the one or more biological reactors can be providedin a system containing modules for effectuating various other processesprior to, concurrent with, or subsequent to the process occurring withinthe cell culture chambers of the biological reactors.

One cartridge can be transferred into harvesting bag or into a newcartridge via sterile welding and sealing. In some embodiments,dendritic cells can be generated and then harvested and cryopreserved,and used on demand. Meanwhile, the system can independently run a secondcartridge for fresh dendritic cell generation.

In certain aspects, computational modeling approaches are used tooptimize the interaction of T-cells with antigen-presenting cells.Computational models in accordance with the present invention take intoaccount the impacts of perfusion and the optimal time required forstimulation, and incorporate both particle interaction-based as well askinetic parameter-based approaches. Example particle interaction-basedand kinetic parameter-based approaches are known in the art, some ofwhich are described herein. For example, with respect to particleinteraction-based approaches, Day and Lythe describe the time requiredfor a T cell to find an APC on the surface of a lymph node using thefollowing expression, where D is the diffusivity of the T cell, and b isthe radius of the APC located centrally within a spherical lymph node ofradius R. See Day et al., Mathematical Models and Immune Cell Biology;2011

$\tau^{\prime} = {{\frac{1}{\frac{4}{3}{\pi\left( {R^{3} - b^{3}} \right)}}{\int_{b}^{R}{4\pi r^{2}{F(r)}{dr}}}} = {\frac{R^{3}}{3{Db}} - {\frac{3}{5}\frac{R^{2}}{D}} + {\frac{2}{3}\frac{b^{2}}{D}} + \ldots}}$

With respect to kinetic parameter-based approaches, Valitutti hasdeveloped a model of the interactions between T-cells andantigen-presenting cells, as shown in FIG. 9 . Valitutti et al., FEB SLett. 2010. However, such interactions have not been modeled within thecontext of a culture chamber or bioreactor.

By incorporating both particle interaction-based as well as kineticparameter-based approaches into the computational models of the presentinvention, automated determination and monitoring of the optimalperfusion rate of a perfusion fluid (e.g., cytokine infused medium) formaximizing the probability of two cell types contacting each otherwithin the cell culture chamber can be achieved.

For example, in certain embodiments, a cell culture system is providedthat includes a cell culture chamber and a central processing unitcomprising memory containing instructions executable by the centralprocessing unit. In certain aspects, the instructions cause the systemto receive as a first input data comprising a size of the cell culturechamber, receive as a second input data comprising a first concentrationof a first cell type and a second concentration of a second cell type inone or more fluids that will be introduced into the cell culturechamber, and calculate, based on the first and second inputs, aperfusion rate of a perfusion fluid that will be introduced into thecell culture chamber that maximizes a probability of the first cell typeand the second cell type contacting each other within the cell culturechamber. Additional details regarding computer systems for implementingthe methods of the present invention within cell culture systems areprovided below.

In some aspects, the system also includes one or more pumps operablycoupled to one or more perfusion fluid reservoirs and operably coupledto the central processing unit, such that the central processing unitalso controls the perfusion rate of the perfusion fluid by controllingthe one or more pumps.

Recycling Medium

Cell culture medium and supplements (such as cytokines) are expensive,and used medium often has residual nutrients in it, which get discarded.Recognizing this, the disclosed systems provide ways to recapture someof the partially used medium and recycle it. In certain methods of theinvention, cells are cultured in one of the disclosed cell culturechambers, wherein cell culture medium is flowed through the cell culturechamber. Generally the fluid flows into an inlet and out of an outlet. Aportion of the cell culture medium that has already flowed through thecell culture chamber and out of the outlet is recycled back into thecell culture chamber during the cell culturing process.

In order to determine how much and/or which portion of used mediumshould be returned to the cell culture chamber, the invention measuresone or more parameters such as nutrient content or pH of the used mediumprior to recycling. The measured nutrients can be glucose, lactate,dissolved oxygen, or cell metabolites. The parameter of interest ismeasured and a processor determines whether the parameter meets apredetermined threshold that indicates that it can be recycled. If so,the medium is sent back into the cell culture chamber.

The used medium can be recycled on its own or it can be combined with abolus of fresh medium. If, on the other hand, the used medium does notmeet the predetermined threshold, it is discarded. In some embodiments avalve operates to direct the used medium either to a waste reservoir orback into the cell culture chamber.

In various embodiments, the recycling can be performed at any frequency.For example, the one or more sensors can check the used medium atregular intervals, such as every second, every minute, every 10 minutes,every hour, etc. In other embodiments, the one or more sensors canoperate continuously by measuring the medium as it goes through a wasteline. In some embodiments, the recycling is controlled through feedbackfrom external filters or sensors that monitor the waste medium todetermine if it can be reused or if it is spent. In some embodiments, anin-line sensor is embedded in the system to monitor waste medium anddetermine if it can be recycled.

Recycling achieves the goal of enabling gas exchange between theexterior of the cell culture chamber and the cells contained withinwhile reducing the amount of medium that would be consumed by theprocess relative to a process where the medium was being perfusedstraight through without recycling.

The rate of recycle can be modulated on the basis of required gasexchange and nutrient supply. For example, in a cell culture processwhere T cells are expanded from a small number to a much larger number,the initial stage of the culture can be carried out at low flow rateperfusion with 100% recycle. This is because the nutrients in the closedperfusion loop are adequate for the small number of cells and the flowrate is set to be sufficient to ensure enough gas exchange (oxygen in,CO2 out). Then, as the cells start to expand, their need for nutrientsand gas exchange grows. Growth is monitored and can form the basis ofdecisions associated with increasing perfusion flow rate (to increasegas exchange) and changing the extent of recycle (recycle only a portionof the medium and add new medium in progressively increasing fractions).In principle, this could be done dynamically and automatically.

In embodiments, the cell culture chamber includes one or more sensorsoperably coupled to the cell culture chamber. The system can beconfigured with various sensor configurations to monitor differentparameters and integrate with the control system. The sensors may becapable of measuring one or more parameters within the cell culturechamber, such as pH, dissolved oxygen, total biomass, cell diameter,glucose concentration, lactate concentration, and cell metaboliteconcentration. The system can be customized with off-the-shelf singleuse sensors for glucose and lactate that sample the perfusion effluentfluid and transmit data. In some embodiments, the cartridges areoptically clear and can be interfaced with sensing modalities such asoptical density or Raman. In some embodiments the sensors may beoperably coupled to a waste line or a waste reservoir, and areconfigured to measure one or more parameters of the fluid that flowstherein. In certain aspects, the one or more sensors are operablycoupled to a computer system having a central processing unit forcarrying out instructions, such that automatic monitoring and adjustmentof parameters is possible. The system may be configured to automaticallyredirect a fluid back into a chamber via an inlet if the fluid meets acertain parameter. In some embodiments, instrumentation interfaces witha control system architecture using computers, networks, and graphicaluser interfaces for process management and other peripheral devices tointerface with process plant machinery. The waste tube may have a valvethat can direct the fluid to one location or another depending onwhether the fluid has a sufficient level of nutrients, for example.Additional details regarding computer systems for implementing methodsof the present invention using the cell culture chambers is providedbelow.

Systems Architecture

Aspects of the present disclosure described herein, such as control ofthe movement of fluid through the system, as described above, and themonitoring and controlling of various parameters, can be performed usingany type of computing device, such as a computer or programmable logiccontroller (PLC), that includes a processor, e.g., a central processingunit, or any combination of computing devices where each device performsat least part of the process or method. In some embodiments, systems andmethods described herein may be performed with a handheld device, e.g.,a smart tablet, a smart phone, or a specialty device produced for thesystem.

Methods of the present disclosure can be performed using software,hardware, firmware, hardwiring, or combinations of any of these.Features implementing functions can also be physically located atvarious positions, including being distributed such that portions offunctions are implemented at different physical locations (e.g., imagingapparatus in one room and host workstation in another, or in separatebuildings, for example, with wireless or wired connections).

Processors suitable for the execution of computer program include, byway of example, both general and special purpose microprocessors, andany one or more processor of any kind of digital computer. Generally, aprocessor will receive instructions and data from a read-only memory ora random access memory or both. Elements of computer are a processor forexecuting instructions and one or more memory devices for storinginstructions and data. Generally, a computer will also include, or beoperatively coupled to receive data from or transfer data to, or both,one or more non-transitory mass storage devices for storing data, e.g.,magnetic, magneto-optical disks, or optical disks. Information carrierssuitable for embodying computer program instructions and data includeall forms of non-volatile memory, including by way of examplesemiconductor memory devices, (e.g., EPROM, EEPROM, solid state drive(SSD), and flash memory devices); magnetic disks, (e.g., internal harddisks or removable disks); magneto-optical disks; and optical disks(e.g., CD and DVD disks). The processor and the memory can besupplemented by, or incorporated in, special purpose logic circuitry.

To provide for interaction with a user, the subject matter describedherein can be implemented on a computer having an I/O device, e.g., aCRT, LCD, LED, or projection device for displaying information to theuser and an input or output device such as a keyboard and a pointingdevice, (e.g., a mouse or a trackball), by which the user can provideinput to the computer. Other kinds of devices can be used to provide forinteraction with a user as well. For example, feedback provided to theuser can be any form of sensory feedback (e.g., visual feedback,auditory feedback, or tactile feedback), and input from the user can bereceived in any form, including acoustic, speech, or tactile input.

The subject matter described herein can be implemented in a computingsystem that includes a back-end component (e.g., a data server), amiddleware component (e.g., an application server), or a front-endcomponent (e.g., a client computer having a graphical user interface ora web browser through which a user can interact with an implementationof the subject matter described herein), or any combination of suchback-end, middleware, and front-end components. The components of thesystem can be interconnected through network by any form or medium ofdigital data communication, e.g., a communication network. Examples ofcommunication networks include cell network (e.g., 3G or 4G), a localarea network (LAN), and a wide area network (WAN), e.g., the Internet.

The subject matter described herein can be implemented as one or morecomputer program products, such as one or more computer programstangibly embodied in an information carrier (e.g., in a non-transitorycomputer-readable medium) for execution by, or to control the operationof, data processing apparatus (e.g., a programmable processor, acomputer, or multiple computers). A computer program (also known as aprogram, software, software application, app, macro, or code) can bewritten in any form of programming language, including compiled orinterpreted languages (e.g., C, C++, Perl), and it can be deployed inany form, including as a stand-alone program or as a module, component,subroutine, or other unit suitable for use in a computing environment.Systems and methods of the invention can include instructions written inany suitable programming language known in the art, including, withoutlimitation, C, C++, Perl, Java, ActiveX, HTML5, Visual Basic, orJavaScript.

A computer program does not necessarily correspond to a file. A programcan be stored in a file or a portion of file that holds other programsor data, in a single file dedicated to the program in question, or inmultiple coordinated files (e.g., files that store one or more modules,sub-programs, or portions of code). A computer program can be deployedto be executed on one computer or on multiple computers at one site ordistributed across multiple sites and interconnected by a communicationnetwork.

A file can be a digital file, for example, stored on a hard drive, SSD,CD, or other tangible, non-transitory medium. A file can be sent fromone device to another over a network (e.g., as packets being sent from aserver to a client, for example, through a Network Interface Card,modem, wireless card, or similar).

Writing a file according to embodiments of the invention involvestransforming a tangible, non-transitory, computer-readable medium, forexample, by adding, removing, or rearranging particles (e.g., with a netcharge or dipole moment into patterns of magnetization by read/writeheads), the patterns then representing new collocations of informationabout objective physical phenomena desired by, and useful to, the user.In some embodiments, writing involves a physical transformation ofmaterial in tangible, non-transitory computer readable media (e.g., withcertain optical properties so that optical read/write devices can thenread the new and useful collocation of information, e.g., burning aCD-ROM). In some embodiments, writing a file includes transforming aphysical flash memory apparatus such as NAND flash memory device andstoring information by transforming physical elements in an array ofmemory cells made from floating-gate transistors. Methods of writing afile are well-known in the art and, for example, can be invoked manuallyor automatically by a program or by a save command from software or awrite command from a programming language.

Suitable computing devices typically include mass memory, at least onegraphical user interface, at least one display device, and typicallyinclude communication between devices. The mass memory illustrates atype of computer-readable media, namely computer storage media. Computerstorage media may include volatile, nonvolatile, removable, andnon-removable media implemented in any method or technology for storageof information, such as computer readable instructions, data structures,program modules, or other data. Examples of computer storage mediainclude RAM, ROM, EEPROM, flash memory, or other memory technology,CD-ROM, digital versatile disks (DVD) or other optical storage, magneticcassettes, magnetic tape, magnetic disk storage or other magneticstorage devices, Radiofrequency Identification tags or chips, or anyother medium which can be used to store the desired information andwhich can be accessed by a computing device.

As one skilled in the art would recognize as necessary or best-suitedfor performance of the methods of the invention, a computer system ormachines employed in embodiments of the invention may include one ormore processors (e.g., a central processing unit (CPU) a graphicsprocessing unit (GPU) or both), a main memory and a static memory, whichcommunicate with each other via a bus.

In an example embodiment shown in FIG. 12 , system 600 can include acomputer 649 (e.g., laptop, desktop, or tablet). The computer 649 may beconfigured to communicate across a network 609. Computer 649 includesone or more processor 659 and memory 663 as well as an input/outputmechanism 654. Where methods of the invention employ a client/serverarchitecture, operations of methods of the invention may be performedusing server 613, which includes one or more of processor 621 and memory629, capable of obtaining data, instructions, etc., or providing resultsvia interface module 625 or providing results as a file 617. Server 613may be engaged over network 609 through computer 649 or terminal 667, orserver 613 may be directly connected to terminal 667, including one ormore processor 675 and memory 679, as well as input/output mechanism671.

System 600 or machines according to example embodiments of the inventionmay further include, for any of I/O 649, 637, or 671 a video displayunit (e.g., a liquid crystal display (LCD) or a cathode ray tube (CRT)).Computer systems or machines according to some embodiments can alsoinclude an alphanumeric input device (e.g., a keyboard), a cursorcontrol device (e.g., a mouse), a disk drive unit, a signal generationdevice (e.g., a speaker), a touchscreen, an accelerometer, a microphone,a cellular radio frequency antenna, and a network interface device,which can be, for example, a network interface card (NIC), Wi-Fi card,or cellular modem.

Memory 663, 679, or 629 according to example embodiments of theinvention can include a machine-readable medium on which is stored oneor more sets of instructions (e.g., software) embodying any one or moreof the methodologies or functions described herein. The software mayalso reside, completely or at least partially, within the main memoryand/or within the processor during execution thereof by the computersystem, the main memory and the processor also constitutingmachine-readable media. The software may further be transmitted orreceived over a network via the network interface device.

INCORPORATION BY REFERENCE

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes.

EQUIVALENTS

While the present invention has been described in conjunction withcertain embodiments, one of ordinary skill, after reading the foregoingspecification, will be able to effect various changes, substitutions ofequivalents, and other alterations to the compositions and methods setforth herein.

1.-12. (canceled)
 13. A method for culturing cells, the methodcomprising: providing a cell culture chamber comprising cells; flowing acell culture medium into the cell culture chamber; removing used cellculture medium from the cell culture chamber; assessing one or moreparameters of the used cell culture medium; and returning the used cellculture medium to the cell culture chamber if the one or more parametersmeets a predetermined threshold.
 14. The method of claim 13, furthercomprising combining the used cell culture medium with a bolus of freshcell culture medium prior to the returning step.
 15. The method of claim13, wherein the assessing step comprises measuring the one or moreparameters using one or more sensors operably coupled to the cellculture chamber.
 16. The method of claim 13, wherein the assessing stepcomprises determining, using a processor, whether the one or moreparameters meets the predetermined threshold.
 17. The method of claim16, wherein the one or more parameters comprises a measuredconcentration of one or more compounds within the used cell culturemedium.
 18. The method of claim 17, wherein the one or more compoundscomprise glucose, lactate, or cell metabolites.
 19. The method of claim13, wherein the returning step comprises redirecting the used cellculture medium from a waste reservoir into the cell culture chamber. 20.The method of claim 13, further comprising: discarding the used cellculture medium if the parameter does not meet the predeterminedthreshold; and flowing fresh cell culture medium into the cell culturechamber.
 21. The method of claim 13, wherein the flowing stepeffectuates at least one of gas exchange and nutrient supply between thecells in the cell culture chamber and the cell culture medium.
 22. Themethod of claim 21, wherein the returning step is based, at least inpart, on at least one of required gas exchange and nutrient supply forthe cell culturing.
 23. The method of claim 19, wherein the assessingstep comprises measuring the one or more parameters using one or moresensors operably associated with the waste reservoir.
 24. The method ofclaim 23, wherein the waste reservoir is in fluid communication with thecell culture chamber.
 25. The method of claim 19, wherein redirectingthe used cell culture medium is performed using a valve operable to flowone or more portions of used cell culture medium back into the cellculture chamber or to the waste reservoir.
 26. The method of claim 13,wherein the one or more parameters are selected from the groupconsisting of pH, dissolved oxygen, nutrient content, total biomass,cell diameter, and cell number.
 27. The method of claim 13, wherein thecell culture chamber is operably connected to one or more pumps.
 28. Themethod of claim 13, wherein the cell culture chamber comprises an inletand an outlet.